| Objective:The PNH cell model was constructed by knocking down the PIG-A gene in THP-1 cell with CRISPR-Cas9 technology,and the PNH animal model was constructed by knocking out the C57BL/6N mouse Pig-A gene with ES targeting technology.By using targeting gene region sequencing technology,some mutated genes in CD59~-cells of PNH patients were found which may related to cloning proliferation.We screened out the mutant gene SUZ12 and explored its role in the pathogenesis of PNH cloning proliferation in the PNH cell model,so as to further understand the pathogenesis of PNH and provide a theoretical basis for exploring new treatment methods.Materials and Methods:1.The PIG-A gene was knocked down in THP-1 cell by using CRISPR-Cas9technology.Verified the successful construction of the cell line by PCR,FCM and WB.The Pig-a gene of hematopoietic tissue was knocked out in C57BL/6N mouse by ES targeting and Cre/lox P technology.Verified the successful construction of the mouse model by PCR,FCM,WB,pathology,and the disease-related indicators of the mouse.2.CD59~+and CD59~-cells from peripheral blood of 23 PNH patients were sorted by FCM,genomic DNA was extracted,and the target region of the candidate gene was sequenced.After filtering and screening the original data,we performed shared mutation,protein interaction,KEGG signaling pathway enrichment and GO annotation analysis.q RT-PCR was used to detect the m RNA expression levels of candidate genes in 20 patients with PNH and 12 healthy controls,and SUZ12 gene was selected for further study.3.qRT-PCR and WB were used to detect SUZ12 m RNA and protein expression level in CD59~-peripheral blood cells of 26 patients with PNH,CD59~+peripheral blood cells of 20 patients with PNH and peripheral blood leukocyte of 23 healthy control.The correlation between the relative expression of SUZ12 in CD59~-cells of PNH patients and the clinical indicators of PNH patients was analyzed.The m RNA and protein expression levels of SUZ12 were detected in the PNH cell model,then knocked down the SUZ12 in THP-1 WT cell and THP-1 KD cell by lentivirus transfection technology.q RT-PCR,WB and FCM were used to verify the knockdown efficiency,and detected the cell proliferation,apoptosis,cell cycle,the percentage of PNH cloning.Results:1.PNH cell model and mouse model construction(1)PCR showed that the target fragment of THP-1 WT cell line could be amplified with the size of 880bp,and the target fragment of THP-1 KD cell line could be amplified with the size of 611bp.WB results showed that the protein level of PIG-A in THP-1 KD cell was significantly reduced.FCM results showed that CD59and Flaer were completely expressed in THP-1 WT cell,the proportion of CD59~-cell in THP-1 KD cell was(97.31±0.8641)%and the expression of Flaer was completely absent.(2)qRT-PCR and WB results showed that the expression level of Pig-A in bone marrow cells of CKO mouse was significantly lower than that of Flox mouse and normal C57BL/6N mouse.The results of FCM showed that glycosylphosphatidylinositol(GPI)and GPI-anchored protein(GPI-AP)were completely expressed in the peripheral blood lines of Flox mouse and normal C57BL/6N mouse,while the GPI and GPI-AP in peripheral blood lines of CKO homozygous mouse were completely deficient.The deficiency proportion of GPI and GPI-AP in peripheral blood cells of CKO heterozygous mouse was slightly different in different cell types.With the prolongation of postnatal time,the deficiency proportion of GPI and GPI-AP in peripheral blood cells of homozygous CKO was stable.The deficiency proportion of peripheral blood cells in CKO heterozygous cells decreased gradually and reached a stable level at about 3 months postnatal.We detected the disease-related indicators in each group and found the CKO mouse had hemocytopenia,hemolysis related indexes and plasma complement C5b-9 level was increased.The above indexes in CKO mouse were statistically different from those in Flox mouse and normal C57BL/6N mouse,suggesting mild hemolysis in CKO mouse.Bone marrow pathology showed that the hematopoietic tissue volume of CKO heterozygous and CKO homozygous mouse(about 95%VOL)was higher than that of normal C57BL/6N mouse and Flox mouse(about 90%VOL).The spleen pathology showed that the spleen of normal C57BL/6N mouse and Flox mouse contained granulosa cells,the granulosa cells of heterozygous CKO mouse were more common,and the granulosa cells of homozygous CKO mouse contained granulosa cells in small clusters and clumps.2.A total of 37,972 SNVs and 26,446 In Del mutation sites were detected by sequencing the target regions of 29 samples from 23 patients with PNH.According to the internationally recognized filtering method,the number of mutation sites was 141SNVs and 66 In Dels.(1)On the basis of filtering harmful loci,there were 11 mutated genes shared by more than 3 patients,including PIG-A,TTN,NCOR2,CPS1,MUC4,SUZ12,LFNG,CELSR2,JAK2,SETBP1 and KMT2D.PIG-A mutations were detected in 18 patients(78.3%),including 16 SNVs and 13 Indels,and exon 2 was the region with the highest mutation frequency.The proportion of MUC4 mutations in patients with thrombus(7/10,70.0%)was significantly higher than that in patients without thrombus(2/13,15.38%),with statistical difference(p=0.0302).(2)GO function of enrichment and enrichment of KEGG results show that the most significant difference in cell components was nuclear lumen,the most significant difference in biological pathways was the synthesis of cell components,the most significant difference in molecular functions was protein binding,and the most significant difference in KEGG pathway enrichment was the tumor-related pathways.The functional interactions of proteins inferred that the proteins that might interact with PIG-A were SUZ12,DHX9,ALPI,CPS1 and NCOR2,and the interaction types were all co-expression.(3)Based on the screening of shared mutated genes,enrichment analysis of candidate genes and protein-protein interaction network screening,we speculated that CPS1,NCOR2,SUZ12,MUC4 and MSH6 might be pathogenic mutated genes associated with disease.q RT-PCR results showed the m RNA expression levels of MUC4 and CPS1 in peripheral blood CD59~-cells of PNH patients were significantly lower than those of healthy controls(p=0.0025,p=0.0351),the m RNA expression level of SUZ12 was significantly higher than that of healthy controls(p=0.0003).The relative expression level of MUC4 in the thrombus group of PNH patients(0.1605±0.1706)was lower than no thrombus group(2.620±3.893)(p=0.0002).3.We selected SUZ12 from the above three genes with different m RNA expression levels to further study its role in PNH cloning proliferation.(1)qRT-PCR was used to detect SUZ12 mRNA expression levels in peripheral blood CD59~+cells and CD59~-cells of PNH patients and peripheral blood leukocytes of healthy controls.The relative expression level of SUZ12 in peripheral blood CD59~-cells of PNH patients(5.119±3.248)was higher than that in CD59~+cells(2.788±1.394)and that in peripheral blood leukocytes of normal controls(2.162±1.948)(p=0.0006).The relative expression of SUZ12 in peripheral blood CD59~-cells of PNH patients was positively correlated with Ret%(r=0.4162,p=0.0385),the proportion of CD59~-erythrocytes(r=0.4636,p=0.0196),the proportion of CD59~-monocytes(r=0.4052,p=0.0495),the proportion of Flaer~-monocytes(r=0.6769,p=0.0004)and the proportion of Flaer~-granulocytes(r=0.6146,p=0.0018).WB showed that the expression level of SUZ12 protein and H3K27me3 methylation in CD59~+and CD59~-peripheral blood cells of PNH patients were higher than that of healthy control,and the expression level of SUZ12 protein and H3K27me3 methylation in CD59~-cells were higher than that of CD59~+cells.The protein expression level of SUZ12 in each group was positively correlated with the methylation level of H3K27me3.(2)The relative expression level of SUZ12 in THP-1 WT cell line(1.214±0.9382)was lower than that of THP-1 KD cell(3.066±1.847),with statistical difference between the two groups(p=0.0188).WB results showed that the protein expression level of SUZ12 and H3K27me3 methylation in THP-1 KD cell were higher than that in THP-1 WT cell,and the protein expression level of SUZ12 was positively correlated with the methylation level of H3K27me3.(3)Lentivirus transfection technique was used to knock down SUZ12 in THP-1WT and THP-1 KD cell,and q RT-PCR verified that the knockdown efficiency of the two cell lines transfected with sh RNA was close to 90%.FCM and WB results inverified the expression level of SUZ12 and the methylation level of H3K27me3 in the sh RNA knockdown group of the two cell lines were significantly lower than that of the empty virus group and the control group.We detected the proliferation activity,apoptosis rates and cell cycle distribution of THP-1 WT cells and THP-1 KD cells at72h after transfection lentivirus.The results showed in sh RNA knockdown group,the proliferation activity decreased,the apoptosis rates increased,and cell cycle arrest occurred in the G0/G1 phase.These changes were more pronounced in THP-1 KD cells than in THP-1 WT.FCM were used to detect the change of CD59~-cell proportion after transfection.The results showed that CD59~+cells were found in all wells of THP-1 WT cells before and after transfection.The proportion of CD59~+cells in THP-1 KD cells was(2.687±0.8641)%before transfection,and the proportion of CD59~+cells in sh RNA knockdown group was(3.047±0.5315)%,(3.397±0.2316)%,(5.130±0.8216)%,(7.627±0.3623)%and(22.75±0.3911)%at 72h,1w,2w,3w and4w after transfection.The proportion of CD59~+cells in sh RNA knockdown group of THP-1 KD cell gradually increased at 3 weeks after transfection,and significantly increased at 4 weeks after transfection.4 weeks after lentivirus transfection,the expression of PIG-A protein in sh RNA knockdown group of THP-1 KD was recovered compared with the empty virus group and the control group.Conclusion:1.THP-1 KD cell line was successfully constructed by knockdown of PIG-A gene in THP-1 WT cell line.The protein level of PIG-A in THP-1 KD cell line was significantly reduced and had GPI and GPI-AP deficiency.The PNH cell model was successfully constructed.Hematopoietic tissue Pig-A gene knockout(CKO)mouse were constructed by ES targeting and Cre/lox P techniques.CKO mouse had mild hemolysis and stable GPI deficiency.2.CD59~+and CD59~-peripheral blood cells of PNH patients were detected by sequencing of target gene regions.CPS1,NCOR2,SUZ12,MUC4 and MSH6 were screened out as disease-related pathogenic mutant genes.The m RNA expression levels of MUC4 and CPS1 in PNH patients were significantly lower than those in the healthy control,and the m RNA expression levels of SUZ12 were significantly higher than that in the healthy control,which proved that the secondary mutant gene may indeed involve in PNH cloning proliferation.3.SUZ12 mRNA,protein expression level and H3K27me3 methylation level in CD59~-peripheral blood cells of PNH patients were higher than those in CD59~+cells of PNH patients and healthy controls.The relative expression level of SUZ12 in CD59~-peripheral blood cells of PNH patients was positively correlated with Ret%and proportion of PNH cloning.These results suggest that SUZ12 may participate in PNH cloning proliferation by regulating H3K27me3.4.The mRNA and protein expression levels of SUZ12 and H3K27me3methylation levels in PNH cell model were higher than those in THP-1 WT,and the protein expression level of SUZ12 was positively proportional to the methylation level of H3K27me3.After knockdown SUZ12 of THP-1 WT and THP-1 KD cell line by lentivirus transfection technique,cell proliferation activity decreased,apoptosis increased,and cell cycle arrest was observed at G0/G1 phase in shrna knockdown group in two cell lines.The effect was more pronounced in THP-1 KD cell lines.The proportion of CD59~+cells in sh RNA knockdown group of THP-1 KD cell line increased gradually at 3 weeks after transfection and increased significantly at 4weeks after transfection.PIG-A protein expression recovered somewhat compared with the empty virus group and the control group.These results suggest that the overexpression of SUZ12 in PNH patients can affect cell proliferation,apoptosis and cell cycle by regulating the level of H3K27me3 methylation,thereby promoting the proliferation of PNH cloning. |
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